Modern agriculture often requires the planting of large numbers of substantially identical plants selected to grow optimally in a particular locale or to possess certain other desirable traits. Production of new plants by sexual reproduction, which yields botanic seeds, is a lengthy, labor-intensive process that is often subject to genetic recombinational events resulting in variable traits in the progeny. Furthermore, inbred strains used to perform such crosses often lack vigor, resulting in low seed productivity.
Botanic seeds, such as those produced by conventional plant breeding, have food-storage organs and protective structures that shelter plant embryos from the harsh soil environment, nurture the embryo during sowing and germination, and enable the seed to survive until conditions are favorable for germination.
In view of the disadvantages of producing large numbers of identical progeny plants by sexual means, propagation of commercially valuable plants via culturing of somatic or zygotic plant embryos has been intensively studied. For some species such "asexual" propagation has been shown to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant. Unfortunately the resulting embryos lack the protective and nutritive structures found in natural botanic seeds. As a result, the embryos are usually cultured under laboratory conditions until they reach an autotrophic "seedling" state characterized by an ability to produce their own food via photosynthesis, resist desiccation, produce roots able to penetrate soil, and fend off soil microorganisms.
Much effort has been directed to the development of techniques for embryogenesis of agronomically important plant species, including conifer species. See, e.g., U.S. Pat. Nos. 4,957,866, 5,034,326, and 5,036,007. Totipotent plant tissue is developed in culture to a stage similar to the natural zygotic embryos occurring in mature seeds. For conifers, these are very small, commonly ranging from about 2-4 mm in length. Embryos have a bipolar form which anticipates the ultimate plant. One end has a latent radicle or root, and the other end has a latent cotyledon and appears similar to a tiny crown.
Somatic embryos lack the endosperm of the natural seed. In order to provide nutrients to the embryo at the time of germination, somatic embryos may be placed on a solid germination medium that contains the necessary carbohydrate and other nutrients, on a growing medium, or on synthetic soil that is saturated with an appropriate nutrient solution. Sterility must be maintained until after the resulting plantlet is well established. Somatic embryos also lack a seed coat and thus are more susceptible than botanic seed to mechanical damage, desiccation, and attack by pathogens and pests.
A preferred method of germinating a unit of totipotent plant tissue, e.g., a plant somatic embryo, is to incorporate it into a manufactured seed (i.e., "artificial seed" or "seed analog"). A number of versions of manufactured seed have been described in the patent literature, including U.S. Pat. Nos. 4,562,663; 4,583,320; 4,615,141; 4,715,143; 4,777,762; 4,779,376; and 4,780,987 and Canadian Patent No. 1,241,552. More advanced versions of manufactured seed that display an improved germination rate are disclosed in U.S. Pat. Nos. 5,427,593 and 5,236,469, incorporated herein by reference.
Methods and apparatus are needed for producing manufactured seed on a commercial scale. If an economical production rate is to be obtained, this process must be automated as much as possible.
One step in this production of manufactured seed is the selection of totipotent plant tissue, e.g., somatic embryos, that are mature enough to incorporate into manufactured seed. There is typically significant variation in morphological normalcy and embryo maturity in somatic embryos produced by conventional tissue culture methods. Manufactured seed containing morphologically abnormal or immature embryos seldom germinate into normal plantlets. Tedious manual selection has been the standard solution to this problem.
Various apparatus have been described for sorting microscopic biological objects such as single cells. See, U.S. Pat. Nos. 3,560,754, 3,710,933, 3,791,517, 3,987,307, and 4,175,662. These apparatus are generally not useful for sorting larger, multicellular biological objects, particularly macroscopic objects such as plant embryos.
A method has been described for separating loblolly pine zygotic embryos and celery somatic embryos according to maturity criteria using sucrose density gradients (Velho et al., HortScience, Programs and Abstracts (suppl.), p. 137, 1989 [Abstract, 87th Annual Meeting of the American Society of Horticultural Science, Tucson, Ariz., Nov. 4-8, 1990]).
U.S. Pat. No. 5,284,765 describes a method of directionally orienting plant embryos in a liquid flotation medium.
Published International Application WO 91/00781 describes the use of a scanner to identify and determine the location of plant embryos and a pipetting mechanism to remove the plant embryos from the liquid culture medium.
Harrell et al., Computers and Electronics in Agriculture 9:13-23, 1993, describes a system for classifying plant embryos. Mature embryos are fixed, manually introduced into the system under non-sterile conditions, and optically imaged. Images of the objects are analyzed using a neural network. Objects identified as mature embryos are deflected out of a gap in a conduit in a medium-filled harvest chamber by an injection of culture medium from a control nozzle and collected. Rejected structures pass through the gap and enter a settlement chamber.
There remains a need for automated apparatus and methods for rapidly and efficiently handling multicellular biological objects such as plant embryos under aseptic conditions without subjecting the objects to mechanical forces that would cause substantial damage. In particular, there is a need for apparatus and methods for rapidly separating embryos that are acceptable for producing manufactured seed from unacceptable embryos and delivering the acceptable embryos in an aseptic fluid stream to a location for incorporation into manufactured seed.